A half-century or more has passed since the creation of a technique of culturing animal cells and the like in a test tube. Such a technique has been significantly developed together with progression of science and technology.
In general, when a living animal is directly used for experiments, the results can be easily understood. However, such direct examination of a living animal has been problematic both technically and economically. Thus, a portion has been excised from an animal, and the cells thereof have been replicated in an artificial environment such as in a Petri dish or in a test tube. This method is called a tissue culture method or a cell culture method. Since such a technique has not been difficult, it has become possible to produce pharmaceuticals, vaccines, diagnostic antigens, etc., by this method. However, in order to culture animal cells in vitro, it is required to culture the cells almost under the same conditions as the original in vivo conditions. For example, conditions, such as an aseptic state or a temperature environment that is set at the same temperature as that in a living body, are applied.
Moreover, even if the aforementioned conditions have been satisfied, it has been necessary for cell division and replication to additionally supply a “cell growth factor” as a nutrient. Examples of such a cell growth factor include various types of hormones, insulin, putrescine, and a fibroblast growth factor. However, such cell growth factors have not yet been clarified in all cell species, and unknown ingredients are included in many cases.
Hence, instead of cell growth factors, animal serums can be used. The effects thereof can be non-specifically anticipated. Among such animal serums, bovine serum is usually selected because of a large bovine population and also because it can be stably supplied. Especially, fetal bovine serum has been frequently used because it contains only a small amount of toxic protein. In scientific studies, there are cases where a bovine-derived protein may be contained in a test material, although bovine is not an animal species of interest. However, the use of such a bovine-derived protein as a pharmaceutical for a human or other animal species may cause a problem.
The first problem is related to allergy. When a vaccine or a drug that contains bovine serum is parenterally injected into a human, or animals, a first injection may not cause a problem in many cases. However, a second injection or later injections may cause a problem regarding an allergy reaction. This phenomenon can be immunologically explained. That is, an animal only slightly reacts with a high-molecular substance (e.g. a protein having a molecular weight of 10,000 daltons or more) when the substance is exposed to the animal for the first time, and thus the administered substance is decomposed in vivo. However, a memory regarding exposure remains in immune system. Accordingly, when the same substance (antigen) is exposed to the animal for the second time or later, immunocytes that memorize the first exposure directly react with the substance, and as a result, a vital reaction that is stronger than that of the first exposure occurs in a short time. Depending on the types of humans or animals, there may be cases where they may have an unfavorable reaction with an antigen that is exposed from the outside. Such a reaction is typically referred to as an allergy reaction. Such an allergy reaction causes fever or swelling at an injected site, and in the worst case, humans or animals die from dyspnea due to respiratory obstruction, collapse, and the like.
The second problem is related to the contamination of pathogens or bovine serum antibodies contained in bovine serum. A famous example is contamination with Pestivirus, Retrovirus, Mycoplasma, etc. of bovine origin. Recently, prion that is a pathogen of bovine spongiform encephalopathy (BSE) known as mad cow disease has become a possible problem.
As stated above, although the use of bovine serum may occasionally cause troublesome occurrences, such bovine serum has commonly been used for the production of vaccines particularly used for a veterinary field that targets animals over the world.
However, an attempt not to use bovine serum in the cell culture method (a serum free medium (SFM) and a serum free cell culture method) and the production of an experimental vaccine for bovines using such a serum free medium and such a serum free cell culture method have currently been reported (Makoschey et al., Serum-free produced bovine herpesvirus type 1 and bovine parainfluenza type 3 virus vaccines are efficacious and safe. Cytotechnology, 39: 139-145, 2002). However, the publication does not disclose a vaccine used for animals other than bovines. Moreover, vaccines used for dogs are not disclosed at all.
Furthermore, it has been reported that MDCK cells had been cultured in a serum free medium and that influenza viruses had been then replicate. (Kessler et al., Suitability of MDCK cells grown in a serum-free medium for influenza virus production. In Brown et al., ed., Inactivated influenza vaccines prepared in cell culture. Dev. Biol. Stand. Basel, Karger, 1999, vol. 98, pp 13-21: Merten et al., Production of influenza virus in serum-free mammalian cell cultures. In Brown et al., ed., Inactivated influenza vaccines prepared in cell culture. Dev. Biol. Stand. Basel, Karger, 1999, vol. 98, pp 23-37: Voten et al., Generation and characterization of reassortant influenza A viruses propagated in serum-free cultured MDCK-SF1 cells. In Brown et al., ed., Inactivated influenza vaccines prepared in cell culture. Dev. Biol. Stand. Basel, Karger, 1999, vol. 98, pp 77-87: Voeten et al., Characterization of high-grown reassortant influenza A viruses generated in MDCK cells cultured in serum-free medium. Vaccine 17: 1942-1950, 1999: Keiichi Makisumi et al., Cells that can be used in serum free culture and suspension culture and method of producing viruses used for vaccines using such cells; domestic re-publication of PCT international publication for patent application (A1); International Publication WO01/064846; International Publication Date: 2001. Sep. 7). However, media disclosed in these publications comprised unknown ingredients, or although they are serum-free, they comprised animal-derived ingredients.
As a simple cryopreservation method of cells cultured in a serum free medium, a method comprising adding 10% of dimethyl sulfoxide (DMSO) to an MDSS2 medium (AXCELL Biotechnologies, F-69610 Saint Genis l'Arentiere) that is one type of SFM, and then cryopreserving Vero cells as monkey kidney-derived cells and BHK-21 cells as hamster kidney-derived cells using the produced medium, has been reported. (Merten et al., A simple serum-free freezing medium for serum-free cultured cells. Biologicals, 23: 185-189, 1995.). However, such an MDSS2 medium comprises 0.3% of peptone derived from casein as an animal protein. In addition, it has also been reported that a medium produced by adding 10% of DMSO to a VP-SFM™ medium (serum-free media) can be applied to cryopreservation of cells that has been cultured in a serum free medium. (Price and Evege, Serum-free medium without animal components for virus production. Focus, 19: 67-69, 1997.). However, the composition of the VP-SFM itself remains unknown.
Thus, it has been desired that a medium that contains no ingredients derived from animals, cells cultured in a medium that contains no ingredients derived from animals, safe vaccines and test reagents, and the like, be developed.